Pantetheine-s-sulfonic acid derivatives and salts thereof

ABSTRACT

PANTEHEINE-S-SULFONIC ACID, 4&#39;&#39;-PHOSPHO-PANTETHEINE-SSULFONIC ACID AND THEIR METAL SALTS WHERE SYNTHESIZED AND FOUND TO BE CLINICALLY EFFECTIVE FOR PROMOTING THE GROWTH OF B. BIFIDUM IN THE INTESTINE OF INFANTS.

April 9, 1974 ZENZQ TAMURA ETAL 3,803,119

PAN'IETHEINE-S-SULFONIC ACID DERIVATIVES AND SALTS THEREOF Original Filed April 20, 1971 CALCIUM D-PANTETHEINE-S-SULFONATE (8mq/kg/d0y) 9- CONTROL PERIOD ADMINISTRATION PERIOD LOG(N) mg Stool FIG. 2

CALCIUM N-PHOSPHO D- PANTHEINE-S-SULFONATE (Bmq/kg/duy) A CONTROL PERIOD ADMINISTRATION 8- PERIOD mg stool 7- United States Patent O US. Cl. 260-112.5 Claims ABSTRACT OF THE DISCLOSURE Pantetheine-S-sulfonic acid, 4'-phospho-pantetheine-S- sulfonic acid and their metal salts were synthesized and found to be clinically effective for promoting the growth of B. bifidum in the intestine of infants.

This is a division of pending application Ser. No. 135,- 640 filed Apr. 20, 1971.

BACKGROUND OF THE INVENTION Field of the invention This invention relates to chemical compounds having the effect of promoting the growth of Bifidobacterium bifiaum (Lactobacillus bifidus) and, more particularly to pantetheine-S-sulfonic acid, 4'-phospho-pantetheine-S-sulfonic acid, their salts, and the methods for preparing the same.

Description of prior art Both the mortality and morbidity of breast-fed infants are about one-half to onethird of those bottle-fed infants, and it has been assumed that B. bzlfidum prevailing in the intestines of breast-fed infants may contribute to their relatively more healthy state.

Therefore, it seems desirable to increase the quantity of B. bifidum in the intestines of infants and several investigations have been directed toward establishing the growth factors of this microbe.

It has been reported that pantetheine, pantethine, or the like have a considerable effect in increasing the B. bifidum in in vitro tests. However, the results of clinical tests using these compounds do not appear to be in accord with the effect accomplished in the in vitro tests. The reason for this disparity between the theoretical and the actual effects has not yet been resolved.

It was first hypothesized that the disparity of results is due mainly to the ingestion of these growth factors through the intestinal wall into the blood on the way to the position of the intestine where B. bifidum resides.

A need exists, therefore, for a compound which would be applicable to clinical use for promoting the growth of Bifidobacteriztm bifidum (hereinafter abberviated to B. bifidum).

SUMMARY OF THE INVENTION One object of this invention is thus to provide a novel compound and a method for synthesizing said compound which would promote the growth of B. bifidum.

This an other objects have now herein been attained on one aspect of the present invention by providing the compound having the following General Formula 1:

wherein Y represents a hydroxy group, a phosphoryl group or a phosphate radical and M represents hydrogen,

ice

an alkali metal atom, an alkaline earth metal atom, or an ammonium group.

The term phosphate radical refers to a radical having the formula OPO- (OM') wherein M represents an alkali metal, an alkaline earth metal or an ammonium group. Examples of M and M are calcium, sodium, barium, potassium, aluminum, etc.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The Compounds I of the present invention have a sulfonic acid radical in its molecule which would be strongly acidic in the intestine when clinically administered. It is believed that this strong acidity in the intestines hinders the intake of these compounds so that they are permitted to reach the microbe colony.

The Compounds I of this invention can be produced by the following method:

wherein Y and M are as defined above.

According to the above reaction scheme, sulfonation takes place on the sulfur atom of pantethine, 4-phosphopantethine or its salt (II) by the action of the sulfite salt or bisulfite salt.

Any sulfite or bisulfite salt can be used for this purpose so long as it can provide sulfite or bisulfite ions to the reaction solution. Suitable such salts include sodium salts, potassium salts, ammonium salts or sulfur dioxide gas introduced in water. The sulfonation reaction is generally conducted in an aqueous solution of (II).

Catalytic metal ions can be used in the reaction solution to complete the reaction smoothly with a high yield of (I). Suitable metal ions include cuprous, cupric, ferrous, ferric, cobaltous, or cobaltic ions. The catalytic ion donor may be a salt which is composed of at least one of these ions and a member selected from the group consisting of sulfuric acid, carbonic acid, hydrochloric acid and sulfurous acid. In addition to these ions, air or oxygen may be introduced into the reaction solution in order to promote the reaction yield to a great extent. The reaction proceeds at temperatures of bet-ween room temperature and 40 C. for from several hours to as much as ten hours, although the exact time is not critical and can be easily ascertained.

The Compounds I thus produced can be easily separated from the reaction mixture by conventional procedures and/or they may be converted to a desirable salt such as a calcium, sodium, aluminum or barium salt by passing an aqueous solution of (I) through a column filled with an ion-exchange resin of desired metal form.

The present compounds show considerable growth activity to B. bz'fidum in in vitro tests. Clinical tests with the Compounds I prove the increase of B. bifidum in the intestine of human infants. The tests were conducted according to the following procedure.

Stool samples were aseptically collected from 8 bottlefed infants before weaning (2 to 4 months of age) during both control and administration periods. (In the control period, the infants were fed with bottled milk, while in the administration period they were fed with milk containing calcium pantetheine-S-sulfonate or calcium 4'-phospho-pantetheine-S-sulfonate in the ratio of 8 mg./ kg. body weight/day.) The stool was collected and suspended in a physiological saline solution in a predetermined dilution. The number of B. bifidum in each stool were determined by culturing the B. bifidum in the suspended solution at 37 C. for 48 to 78 hours in a Negishi medium forB. bifidum under anaerobic condition, and checking the colonies number.

The experimental results are shown in FIGS. 1 and 2, wherein the number of B. br'fidum per mg. of the stool is plotted on the vertical axis on a logarithmic scale.

Another desirable eflfect on the infants has been observed during the administration period; for example, it was found that Compound I caused a distinguishable increase in body weight, increase of the reticulocyte, increase of the soterocyte, and decrease in the blood level of cholesterols and nitrogen value due to urea.

As described above the Compounds I are a useful growth factor for B. bifidum in even clinical administration, and furthermore the method of preparation has a great advantage in providing a high yield.

Having now generally described the invention, a more complete understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.

EXAMPLE 1 In 100 ml. of 0.2 M. ammonia solution containing 0.2 M. of sodium sulfite, 555 mg. of pantetheine was dissolved. The solution was kept at room temperature for 22 hours and air was introduced. The reaction solution was concentrated to dryness in vacuo. The residue was extracted with methanol and methanol extracts were concentrated to dryness in vacuo. The residue dissolved in water was applied to the column filled with QAE- Sephadex (CO -Form, 1.5 x 19' cm.). The column was washed with water and eluted with 500 ml. of water and a solution of 500 ml. of 0.5 M. of NH4HCO'3 according to the linear gradient elution method. The eluate was collected by 50 ml. of fraction. The fractions of No. 5 and No. 6 were combined and concentrated to dryness in vacuo. The aqueous solution of the residue was passed through 40 ml. of the column filled with SE-Sephadex (CA-Form) and the column was eluted with water. The eluate was concentrated to drynesss and the residue was dissolved in methanol.

Insoluble substances in the methanol solution were filtered off and the methanol was removed from the filtrate. The aqueous solution of the residue was concentrated to dryness.

The residue was dried in vacuo at 40 C. to give 213 mg. of calcium pantetheine-S-sulfonate having a melting point of 174 C. (decomp.).

[od 13.9 (1.95% :H O)

Elemental analyses for C H 0 N S Ca.Calcd. (percent): C, 35.00; H, 5.59; N, 7.38. Found (percent): C, 34.85; H, 5.73; N, 7.09.

EXAMPLE 2 In 60 ml. of water, 8.05 g. of ammonium sulfite, 5.55 g. of pantethine and 500 mg. of copper sulfate were in order dissolved. 15 ml. of methanol was then added. The solution was allowed to stand in air overnight. The reaction solution was concentrated to dryness in vacuo and the residue was dissolved in methanol.

After filtering oil? the insoluble substance, the filtrate was concentrated to dryness. The aqueous solution of the residue was applied to the column of Amberlite 120 (Ca- Form) and eluted with water. After the eluate was concentrated to dryness, the residue was dissolved in methanol and the insoluble substances were removed from the solution. Concentration of the aqueous solution of the residue yielded 7.2 g. of calcium pantetheine-S-sulfonate in powder form. The physical properties of this compound were found to be almost identical with hose described in Example l. The aqueous solution of the calcium salt obtained by the method described above was passed through a column of Amberlite IR-120 (H-Form) and eluted with water. Rapid concentration of the eluate to dryness at low temperature yielded a colorless powder of pantetheine- S-sulfonic acid.

Elemental analyses for C H O N S H O.Calcd. (percent): C, 35.07; H, 6.43; N, 7.44. Found (percent): C. 35.00; H, 6.16; N, 7.70.

EXAMPLE 3 The procedure of Example 2 was repeated using the barium form of the resin instead of IR-lZO (Ca-Form). The eluate was concentrated to dryness and the residue was extracted with methanol. Addition of acetone to the methanol solution yielded 8.1 g. of barium pantetheine-S- sulfonate.

Elemental analyses for Calcd. (percent): C, 32.98; H, 5.32; N, 6.16. Found (percent): C, 33.39; H, 5.55; N, 6.77.

EXAMPLE 4 In 25 ml. of an aqueous solution containing 3 g. of sodium sulfite, a mixture of 1.1 of pantethine and 30 mg. of copper sulfate were dissolved. To the solution, 6 ml. of methanol was added and then air was introduced overnight. The reaction solution was concentrated to dryness in vacuo. The residue was extracted with methanol and the methanol solution was concentrated to dryness in vacuo.

The residue was dissolved in ethanol and to the solution, ether was added. The resulting precipitate was collected by filtration and dissolved in water. The residue obtained by concentration of the aqueous solution in vacuo was dried in vauo to give 1.4 of sodium pantetheine- S-sulfonate.

Elemental analyses for C H O N S Na. /2H O.- Calcd. (percent): C, 33.92; H, 5.69; N, 7.19; Na, 5.91. Found (percent): C, 33.98; H, 5.47; N, 6.93; Na, 6.06.

EXAMPLE 5 33 e3 21 e s s a Calcd. (percent): C, 37.36; H, 6.01; N, 7.26. Found (percent): C, 37.85; H, 6.26; N, 7.25.

EXAMPLE 6 In a mixture of 20 m1. of water and 5 ml. of methanol, 1.11 g. of pantethine, 1.25 g. of sodium hydrogen sulfite and 200 mg. of copper sulfate were dissolved. Air was introduced into the solution overnight. After neutralization with sodium hydroxide, the solution was concentrated to dryness in vacuo. The residue was extracted with methanol to give sodium pantetheine-S-sulfonatc. The aqueous solution of the sodium salt was passed through a column of Amberlite 111-120 (Ca-Form) and eluted with water. 7

The eluate was concentrated to dryness in vacuo to give 1.3 g. of calcium pantetheine-S-sulfonate.

EXAMPLE 7 In 30 ml. of water, 1.11 g. of pentethine, 2.1 g. of ammonium sulfite and mg. of ferric sulfate were dissolved. Air was introduced into the solution for 48 hours.

Insoluble substances were filtered off and the filtrate was concentrated to dryness in vacuo. The residue was extracted with methanol and from the methanol solution, methanol was removed in vacuo. Treatment of the residue in a manner similar to that of Example 2 yielded 935 mg. of calcium pantetheine-S-sulfonate.

EXAMPLE 8 In 30 ml. of water, 1.11 g. of pantethine, 1.6 g. of ammonium sulfite and 100 mg. of cobalt carbonate were dissolved. Air was introduced into the solution for 48 hours. The reaction solution was concentrated to dryness in vacuo and the residue was extracted with methanol. After decolorizing the methanol solution with charcoal, methanol was removed from the filtrate. Treatment of the residue in a manner to that of Example 2 yielded 990 mg. of calcium pantetheine-S-sulfonate.

EXAMPLE 9 In 20 ml. of water, 1.1 g. of pentethine and 2.1 g. of ammonium sulfite were dissolved and the solution was allowed to stand at room temperature overnight. The reaction solution was concentrated to dryness in vacuo and the residue was extracted with methanol. From the methanol extracts, methanol was distilled off. The aqueous solution of the residue was passed through a column of Amberlite IR-120 (Ba-Form) and eluted with water. The eluate was concentrated to dryness in vacuo. Addition of isopropanol to the residue dissolved in a small amount of methanol yielded 0.8 g. of barium pantetheine-S-sulfomate in powder form. The physical properties of this compound were found to be identical with those described in Example 3.

EXAMPLE 10 About 1.73 g. of 4'-phosphopantethine calcium salt was dissolved in 20 ml. of water. Into the solution, 20 ml. of aqueous solution containing 50 mg. of copper sulfate were added. Air was introduced into the solution overnight. The resulting precipitate was filtered oil and the filtrate was concentrated to dryness in vacuo. The residue was dissolved in water. The solution was applied to a column of Amberlite IR-120 (H-Form) and the column was eluted with water. The water eluate was neutralized with aqueous calcium hydroxide solution and concentrated in vacuo. The residue was dissolved in a small amount of water and the insoluble substance was filtered off. The filtrate was concentrated to dryness in vacuo. The residue was dried with phosphoric anhydride in vacuo to give 1.8 g. of calcium 4'-phosphopantetheine-S-sulfonate.

0. +7.2 o=1.9s, H2O) Elemental analyses for C H O N P- S Ca .--Calcd. (percent): C, 26.66; H, 4.08; N, 5.65. Found (percent): C, 26.61; H, 4.08; N, 5.53.

The calcium salt thus obtained was subject to the column of IR-l20 (H-Form) and eluted with water. The eluate was concentrated under the reduced pressure to give 4'-phosphopanthetheine-S-sulfonic acid.

Elemental analyses for C H N PS :2H O.-Calcd. (percent): C, 27.84; H, 5.74; N, 5.90. Found (percent): C, 28.28; H, 5.60; N, 6.20.

EXAMPLE 11 In a manner similar to that of Example 10, by using barium hydroxide instead of calcium hydroxide, 2.3 g. of barium 4'-phosphopantetheine-S-stflfonate was obtained.

Elemental analyses for C I-I O N S Ba '2H O.- Calcd. (percent): C, 20.04; H, 3.36; N, 4.25. Found (percent): C, 20.11; H, 3.58; N, 3.98.

EXAMPLE 12 About 431 mg. of calcium 4-phosphopantetheine was dissolved in ml. of water. Into the solution, an aqueous solution containing 504 mg. of sodium sulfite and then an aqueous solution containing 20 mg. of copper sulfate were added. Air was introduced into the solution overnight. The precipitate was filtered ofi. The filtrate wasconcentrated to dryness in vacuo and the residue was extracted with methanol; then, the methanol was removed from the methanol solution. The aqueous solution of the residue was applied to a column of Amberlite IR-120 (Na-Form) and eluted with water. Concentration of the eluate to dryness under the reduced pressure yielded 490 mg. of the sodium 4-phosphopantetheine-S-sulfonate in powder form.

Elemental analyses for C H O N PS- Na -2H O. Calcd. (percent): C, 24.45; H, 4.48; N, 5.18. Found (percent): C, 24.50; H, 4.60; N, 4.90.

EXAMPLE 13 In a manner similar to that of the foregoing Example 12, using the column of IR-l20 (Al-Form) instead of IR-120 (Na-Form) yielded 420 mg. of aluminum 4-phosphopantetheine-S-sulfonate.

Elemental analyses for C H O N PS Al-3H O.- Calcd. (percent): C, 25.58; H, 5.08; N, 5.43. Found (percent): C, 25.56; H, 5.10; N, 5.30.

EXAMPLE 14 About 431 mg. of 4-phosphopantethine calcium salt was dissolved in 5 ml. of water. Into the solution, an aqueous solution containing 833 mg. of sodium hydrogen sulfite was added and followed with addition of an aqueous solution containing 20 ml. of copper sulfate. Air was introduced into the solution overnight. In a manner similar to that of Example 10, 445 mg. calcium 4'-phosphopantetheine-S-sulfonate was obtained.

EXAMPLE 15 In 15 ml. of water, 431 mg. of 4'-phosphopantethine calcium salt, 670 mg. of ammonium sulfite and 50 mg. of ferrous sulfate were dissolved and air was introduced into the solution overnight. The reaction solution was treated in a manner similar to that of Example 10 to give 450 mg. of calcium 4'-phosphopantetheine-S-sulfonate.

EXAMPLE 16 In an aqueous solution containing 0.2 M. of ammonia and 0.2 M. of ammonium sulfite, 431 mg. of 4'-phosphopantethine calcium salt was dissolved and the solution was allowed to stand for 22 hours at room temperature. Insoluble substances were filtered off and the filtrate was concentrated to dryness in vacuo. The aqueous solution of the residue was subjected to the column (1.5 x 19 cm.) filled with QAE-Sephadex (OH-Form) and the column was washed with water and eluted with 500 ml. of water and a solution of 500 ml. of 0.5 M. of NH HCO according to the linear gradient elution method. The eluate was collected by 50 ml. of fraction. The fractions of No. 9- No. 11 were concentrated to dryness in vacuo. The aqueous solution of the residue was subjected to the column of 40 ml. of SE-Sephadex (Ca-Form) and eluted with water. The eluate was concentrated to dryness in vacuo and the residue was dissolved in a small amount of water. Insoluble substances were filtered off.

Concentration of the filtrate to dryness in vacuo yielded 283 mg. of calcium 4'-phosphopantetheine-S-sulfonate. [atJ +7.2( 1.94%: H O) M.P.: higher than 300 C.

Having now fully described the invention, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention.

Accordingly, what is claimed as new and desired to be secured by Letters Patent of the United States is:

1. A compound of the formula:

wherein Y represents a member selected from the group consisting of a phosphoryl group and a phosphate radical, and M represents a member selected from the group consisting of hydrogen, an alkali metal atom, an alkaline earth metal atom and an ammonium group.

2. The compound of claim 1, which is selected from the group consisting of 4'-phospho-pantetheine-S-sulfonic acid and the metal salts thereof.

References Cited UNITED STATES PATENTS 3,413,330 11/1968 Westland L 260453 FOREIGN PATENTS 1,41 8,664 12/ 1964 France.

US. Cl. X.R. 

